In molecular biology experiments, a gene carrier called “vector” is used for convenience in dealing with genes and for long term preservation. This vector is basically circular double-stranded DNA (dsDNA). According to the elements and use, the vector is classified into cloning vector, transcription vector and translation vector, etc.
When a gene is inserted into a vector, it is called “cloning”. Various methods have been developed for cloning. Among them, the most common method is to use restriction enzyme treatment and ligation. In restriction enzyme treatment, restriction enzymes digest DNA into DNA fragments. In ligation, a ligase can insert the purified DNA fragment generated by precedent restriction enzyme treatment into a vector.
In this method, restriction enzymes digest DNA in the specific site which an experimenter wants to cut and ligase mediates ligation between ends of DNA fragments. In this method, DNA fragment is first prepared by digesting DNA using restriction enzymes and the vector is also digested by the same restriction enzyme to make DNA cloning site (sometimes, DNA and a vector are digested with different enzymes, which is not general, though). When DNA is digested with a restriction enzyme, a specific site of DNA cut by the restriction enzyme is called “restriction enzyme recognition sequence”. This restriction enzyme recognition sequence is also called “restriction enzyme recognition site” and in some cases it is understood as a restriction enzyme catalytic site (cleavage site). This restriction enzyme recognition sequence differs among restriction enzymes. So, an experimenter needs to select a proper restriction enzyme for his experiment. DNA digested with a restriction enzyme has sticky-end or blunt-end.
To prepare a DNA fragment to be inserted into a vector, DNA is treated with restriction enzymes. The DNA fragment is then purified from restriction enzyme treated reaction mixture. To prepare a vector fragment, a vector is also treated with restriction enzymes and as a result a vector fragment having DNA cloning site is prepared (linearized structure). This vector fragment is also purified from restriction enzyme treated reaction mixture. The purification of DNA fragment and vector fragment from restriction enzyme treated reaction mixture can be performed by using a commercial DNA purification kit. And this purification can be performed easily by those in the art, so that precise description is not given herein.
To ligate a purified DNA fragment and a purified vector fragment, these two fragments are added to ligation buffer at a proper molar ratio, to which ligase is added, followed by inducing ligase reaction (ligation among DNA fragments). Accordingly, the end of DNA fragment is linked to the end of linearized vector fragment, resulting in a circular plasmid, suggesting the completion of cloning. The plasmid herein indicates the circular double-stranded DNA.
It is not easy and inefficient to separate and purify a cloning product alone from ligation reaction mixture to use it for the next step of experiment. Even if it is successfully separated and purified, the amount is not sufficient for the next step. Therefore, in most cases, transformation using Escherichia coli is performed to obtain enough amount of a cloning product. In general, transformation is performed by using heat-shock method. Sometimes, transformation using electroporation is performed but to transform E. coli using a small sized plasmid, heat-shock method is enough working. Transformation by heat-shock is performed as follows. 10 μl of ligation mixture was added to 100 μl of E. coli solution in which cell membranes of the cells are loosened by treating calcium chloride (generally called ‘competent cell’). The mixture is then left in ice for 30 minutes to let the cloning product to be adhered on the surface of E. coli. 30 minutes later, the mixture is transferred quickly to a 42° C. bath and left therein for 1 minute. This is called as ‘heat-shock’. At this time, the cloning product penetrates into E. coli. After heat-shock, the mixture is transferred again into ice quickly and left therein for 1 minute. After 1 minute of staying in ice, the mixture is taken out of ice, to which 800 μl of a proper culture medium is added. Generally, LB medium (trypton, 10 g/L; yeast extract, 5 g/L; sodium chloride, 10 g/L) is used in transformation. After adding a culture medium, the reaction mixture is incubated at 37° C. for one hour. During this incubation, cell membranes damaged by calcium chloride treatment become recovered. After one hour of incubation, the mixture is spread on a plate medium containing a proper antibiotic, followed by culture in an incubator until proper sized colonies are formed. At this time, a colony indicates E. coli cells proliferated from one E. coli cell. When colonies are formed big enough, it is considered that transformants are generated. And, the transformant is transferred into a liquid medium (generally LB medium), followed by further culture. From the cultured cells, enough amount of plasmid can be obtained.
To increase convenience and efficiency of cloning, diverse methods have been developed by modifying the conventional cloning method composed of a restriction enzyme treatment and ligation. One example is the method of using a specially designed vector capable of accepting a PCR-amplified product, which is related to the present invention.
PCR is a reaction using DNA polymerase, which produces massive amount of DNA amplicons from template DNA. PCR is a well-known, general technique used for biological study and for disease diagnosis. Cloning of such PCR product can also be achieved by the cloning method using a restriction enzyme and ligase as explained hereinbefore. However, to do so, an amplified PCR product has to contain a restriction enzyme recognition sequence. To introduce a restriction enzyme recognition sequence into a PCR product, a specifically designed PCR primer is required. The primer has to contain 6-10 nucleotides corresponding to a restriction enzyme recognition sequence selected by an experimenter in addition to the nucleotide sequence (priming sequence) binding to a template. A PCR product obtained by using a primer containing a selected restriction enzyme recognition sequence cannot be directly linked to a vector fragment because the cloning site is not exposed. So, after digesting a PCR product by a corresponding restriction enzyme and purification of the restriction enzyme treated PCR product, insertion into a vector fragment is performed.
However, the above method uses a PCR product as a cloning target instead of general DNA, which is the only difference from the conventional cloning method composed of a restriction enzyme treatment and ligation. Therefore, the method does not provide a special effect or convenience. Besides, a primer specially designed as the above contains additional nucleotides in addition to annealing sequence binding to nucleotide sequence of a target gene, suggesting that efficiency in template specific primer annealing decreases. A PCR product generated by the method contains restriction enzyme recognition sequences in both ends, suggesting that digestion efficiency by a restriction enzyme decreases. In general, when a restriction enzyme recognition sequence is located in terminal region of DNA molecule, digestion efficiency is lower than when it is located in the middle.
Therefore, many researchers tried to develop more simple method for cloning of PCR product and to improve efficiency of restriction enzyme treatment to a PCR product. As a result, they proposed the method in which restriction enzyme treatment step that has been always necessary according to the conventional cloning method is omitted. As an example, PCR product cloning method using topoisomerase I or a vector designed to be suitable for direct cloning of PCR product without restriction enzyme treatment was proposed.
The PCR product cloning method of the present invention is related to the latter, which is direct cloning of PCR product into a linearized vector fragment without restriction enzyme treatment. The linearized vector used for direct PCR product cloning without using a restriction enzyme is largely divided into two groups according to the type of terminal region of the PCR product used in cloning. One is the linearized vector fragment designed suitable for direct cloning of PCR product having blunt ends. The other is the linearized vector fragment designed suitable for direct cloning of PCR product having non-complementary unpaired single overhang in the end of only one chain of double strands.
A PCR product is a double-stranded DNA having a linearized structure in which two strands are complementarily combined. In general, the end form of PCR product is determined by DNA polymerase used in its amplification. That is, a PCR product can have either blunt ends or non-complementary unpaired single overhang ends according to the kind of DNA polymerase used. So, an experimenter can choose proper DNA polymerase considering the purpose of the experiment. For example, when Taq DNA polymerase frequently used for PCR as a thermophilic enzyme or Tth DNA polymerase is used, a unique PCR product having non-complementary unpaired single overhang at both 3′-ends is produced by template-independent addition. The nucleotide residue forming the non-complementary unpaired single overhang can be any of single deoxyadenosine monophosphate residue (dAMP; indicated as ‘A’ herein for convenience), single deoxythymidine monophosphate residue (dTMP; indicated as ‘T’ herein for convenience), single deoxyguanosine monophosphate (dGMP; indicated as ‘G’ herein for convenience), and single deoxycytidine monophosphate residue (dCMP; indicated as ‘C’ herein for convenience). Among them, the most frequent occurrence is A-overhang. It was reported that the type of overhang is determined by primer sequence (Hu, DNA Cell Biol. 12: 763, 1993; Magnuson et al., BioTechniques 21: 700, 1996).
As explained hereinbefore, a specially designed vector (mentioned as a vector for convenience, but exactly speaking it is a vector fragment) is required for direct cloning of a PCR product having blunt ends or overhang ends.
Basically, cloning of a PCR product having blunt ends (or even if a PCR product had overhang ends but the overhang ends have been eliminated by the treatment of nuclease, it is included in the group of PCR products having blunt ends) can be achieved only with a linearized vector fragment having blunt ends. So, this kind of cloning is theoretically simple. To prepare a linearized vector fragment having blunt ends, a restriction enzyme such as EcoR V, Dra I, or Hinc II can be used. A PCR product having blunt ends can be linked to a linearized vector fragment having blunt ends by ligase under the conventional DNA ligation conditions. However, according to this method, coupling between vector fragments or intra-molecularar circulization of vector fragments is dominant over real cloning, suggesting that cloning efficiency is reduced.
On the other hand, the production of a vector fragment for the direct cloning of such a PCR product having unpaired single overhang and the cloning of the same are rather complicated. For direct cloning of a PCR product having overhang ends, a linearized vector fragment having overhang ends matching to the overhang ends of a PCR product (complementary binding) is required. So, the overhang of a linearized vector fragment has to be located in the opposite strand to the overhang of a PCR product for complementary binding. To help understand the relative location of overhang, a schematic diagram illustrating the relative locations of terminal overhangs of a vector fragment and a PCR product is presented in FIG. 1. In PCR products, A-overhang is most frequently observed. So, T-overhang is most appropriate as terminal overhang of a vector fragment, but not necessarily limited thereto. Instead, a vector fragment can have any of A-overhang, C-overhang, and G-overhang. It is general to unite two overhangs of both ends of a vector fragment, but not always limited thereto. However, since A-overhang is most frequently observed in end of a PCR product, it is preferred for a vector fragment to have T-overhangs in both ends for complementary binding. Therefore, description hereinafter is focused on the vector fragment having T-overhang. It is only for convenience in explanation but cannot limit the present invention thereto.
Linearized vector fragments having overhang ends can be produced by two different methods. Both use a circular parental vector (indicating a starting vector for the production of a vector fragment, and in this invention, it is called as ‘parental vector’). One of the methods is the method using restriction enzymes capable of producing blunt ends. In this method, a circular parental vector is digested with a restriction enzyme capable of producing blunt ends. Then, the prepared linearized vector fragment having blunt ends is purified. In the next stage, an overhang is added to the blunt ends of the linearized vector fragment, followed by purification again. At this time, a restriction enzyme capable of producing blunt ends is exemplified by EcoR V, Dra I, and Hinc II. To form an additional overhang, Taq DNA polymerase (Marchunk, D. et al., Nucleic Acid Res. 19: 1154. 1991) or terminal deoxynucleotidyl transferase (Holton, T. A. et al., Nucleic Acid Res. 19: 1156, 1991) is used in the presence of excess amount of Mg2+. According to this method, efficiency of addition of overhang to the end of a vector fragment depends on the activity of the selected modifying enzyme. So, there is still a high chance for a vector fragment to remain as an incomplete vector fragment that does not contain an additional overhang. Such an incomplete vector fragment without having an overhang can form intra-molecular circulizated molecule (self-ligation) during ligation and cannot be used for cloning. Therefore, this causes the decrease of cloning efficiency.
The other method is the method of using restriction enzymes capable of producing a linearized vector fragment having overhang ends without any additional treatment (Yoshikazu, I. et al., Gene 130: 152, 1993; David A. M., et al., Bio/Technology 9: 65, 1991). A restriction enzyme used at this time is exemplified by Ahd I, BciV I, Bfi I, Bfu I, Bmr I, BspOV I, Dri I, Eam1105 I, EclHK I, Hph I, Mbo I, Mnl I, Ncu I, NruG I, Taa I, Tsp4C I and Xcm I. Particularly, a parental vector having 1-2 restriction enzyme recognition sequences capable of being used for producing overhang ends directly is treated with a proper restriction enzyme, and as a result, a vector fragment having non-complementary unpaired single overhang at the end is prepared. It is not preferred but is possible for a parental vector to have several restriction enzyme recognition sequences capable of being used for producing overhang ends in several different regions. If that is the case, effective restriction enzyme recognition sequences are two sequences that are located outmost from the DNA fragment cut out of the parental vector.
To perform the cloning of a PCR product having overhang ends into a linearized vector fragment having overhang ends, these two fragments and ligase are added to ligation buffer, followed by ligation. To obtain massive cloning products after ligation, E. coli is transformed with the ligation mixture. At this time, other plasmids effective in transformation can be included in addition to the cloned plasmid in ligation mixture. The parental vector used for the production of a linearized vector fragment having overhang ends or the recirculized plasmid of the incompletely digested parental vector (one of the two restriction enzyme recognition sequences is digested but recirculized as a parental vector from ligation) can be included, although the amount is small. It is theoretically possible to eliminate these vectors completely by intensive separation and purification after restriction enzyme treatment. However, such complete elimination is not easy and some of the vectors are included generally. When a parental vector is digested with a restriction enzyme to produce a linearized vector fragment having overhang ends, a released DNA fragment is too small. So, on agarose gel electrophoresis, difference in mobility of each the vector digested completely and the vector digested incompletely is not so great. Thus, no matter how badly want to separate and purify a completely digested vector fragment alone using gel-extraction, an incompletely digested vector fragment or non-digested parental vector can still be included, which has to be overcome.
For the conventional transformation, a selection medium containing antibiotics is generally used. So, E. coli that does not have such a plasmid containing an antibiotic resistant gene cannot be growing on that medium. E. coli that has not been transformed with the plasmid containing an antibiotic resistant gene is fundamentally eliminated from the selection. Even if such E. coli that is not transformed with the plasmid containing an antibiotic resistant gene in the selection medium is eliminated first, E. coli transformed with a recirculized plasmid generated from a incompletely digested parental vector fragment, a parental vector itself, or a cloning product can have the plasmid containing an antibiotic resistant gene, suggesting that the E. coli transformed with these plasmids can grow in the selection medium containing antibiotics. So, the selection of the transformant having a cloning product using antibiotics is not possible.
To isolate only the transformant having a cloning product, blue/white colony selection method is proposed. The blue/white colony selection method uses a special medium and E. coli transformed with a proper cloning product can be determined by its apparent color. Accordingly, it is possible to distinguish a colony having a plasmid successfully cloned from a colony which does not. E. coli used for blue/white colony selection is genetically modified to fit for blue/white selection method, which is well known to those in the art, so the explanation thereof is omitted herein. If E. coli does not have a plasmid or has a plasmid but not expressing alpha-peptide, a white colony is formed in blue/white colony selection. E. coli which does not have any plasmid cannot be growing in the selection medium containing antibiotics because it does not contain any antibiotic resistant gene. So, there is no need to worry about such E. coli. In general, a transformant having a recirculized plasmid generated from an incompletely digested parental vector fragment or a parental vector appears as a blue colony in blue/white colony selection, while a transformant having a cloning product appears as a white colony in blue/white colony selection. That is, E. coli having the unproper cloning product can express alpha-peptide and appears a blue colony. If E. coli is transformed with a proper cloning product, E. coli cannot express alpha-peptide and appears as a white colony.
Blue/white colony selection is based on the action of alpha-peptide. So a plasmid applicable for blue/white colony selection has to contain a gene encoding alpha-peptide. Also, factors and sequences necessary for the expression of alpha-peptide (corresponding promoter, etc) have to be included in the plasmid as well. Alpha-peptide is N-terminal region of beta-galactosidase and the gene encoding alpha-peptide is represented by SEQ. ID. NO: 1. Alpha-peptide is an enzyme converting 5-Bromo-4-chloro-3-indolyl β-D-galatopyranoside (X-Gal) into a blue-colored material. To perform blue/white colony selection, alpha-peptide has to be expressed from the plasmid used for the transformation. To do so, isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to the medium as expression inducer.
Therefore, alpha-peptide gene sequence has to be included in a plasmid used for the transformation for blue/white colony selection and the alpha-peptide sequence included in plasmid is provided by a cloning vector used in construction of the plasmid. Some parts of alpha-peptide sequence can be modified. In many commercial cloning vectors, some parts of alpha-peptide sequence have been modified from an original sequence to introduce cloning region within the alpha-peptide gene. Such modification is of course limited and has to be confirmed by experiments. Examples of acceptable modification have been known, based on which diverse modification attempts in the sequence have been made. Relevant references are easily obtained by those in the art, so the precise explanation is omitted in this invention.
In order for a transformant having a recirculized plasmid generated from an incompletely digested parental vector fragment or a parental vector to form a blue colony, alpha-peptide has to be expressed from these plasmids. To do so, reading frame of alpha-peptide is composed of a contiguous and non-overlapping set of three-nucleotide codons. Even if a part of alpha-peptide sequence is modified, complete open reading frame has to be in-frame. In order for a transformant to form a white colony, the cloned gene is located in the inside of alpha-peptide gene. That is, the cloning has to be able to knockout the alpha-peptide gene. In general, when a big size gene is inserted within alpha-peptide gene, alpha-peptide gene is knockout. So, blue/white colony selection explained above has been widely used to isolate colonies having cloning products.
As explained hereinbefore, distinguishment of a parental vector and an incompletely digested parental vector fragment from a completely digested parental vector is very difficult. They are usually mixed. Precisely, a transformant having a parental vector, a transformant having a recirculized plasmid, and a transformant having a cloning product can be distinguished in blue/white colony selection, so it causes no big confusion. Significant problem caused by inefficiency of a restriction enzyme is that the amount of effective vector fragments obtainable from equal amount of a parental vector is much less, resulting in inefficiency in production process. If cloning is performed with such vector fragments including a parental vector or an incompletely digested parental vector, which means as the amount of effective vector fragment used for real cloning becomes less, cloning efficiency will be decreased. Even if it is so difficult to distinguish a completely digested parental vector fragment alone from an incompletely digested parental vector fragment or a parental vector, the problem can be minimized by maximizing efficiency of a restriction enzyme during the production of a vector fragment. If the distance between restriction enzyme recognition sequences is too close, the functions of those restriction enzymes are interrupted, resulting in incomplete digestion. This problem is only caused by too close distance between restriction enzyme recognition sequences in the parental vector used for vector fragment production, so it can be improved by extending the distance. And the extension of the distance between restriction enzyme recognition sequences in the parental vector can be achieved by inserting an additional gene in between the restriction enzyme recognition sequences. The insertion of an additional gene can increase efficiency of a restriction enzyme and facilitates separation and recovery of products by taking advantage of difference in size of products after the treatment of restriction enzyme. However, such insertion of an additional gene in between the restriction enzyme recognition sequences present within the alpha-peptide gene may knockout alpha-peptide gene necessary for blue/white colony selection. This is consistent with that a transformant transformed with a cloning product having gene insertion in alpha-peptide sequence appears as a white colony in blue/white colony selection. That is, the insertion of an additional gene in between the restriction enzyme recognition sequences brings another problem of making selection of colonies having a parental vector or a recirculized parental vector by color impossible because all transformants having a cloning product and a parental vector appear as white colonies in blue/white colony selection.
The present inventors completed this invention by establishing a method for producing a PCR product cloning vector fragment which can overcome the problems of the conventional method such as inefficient digestion in restriction enzyme treatment and difficulty in separation of a restriction enzyme treated vector fragment and at the same time facilitates distinguishment of each transformant respectively transformed with a parental vector and a recirculized plasmid generated from an incompletely digested parental vector fragment by making them presented as blue colonies in blue/white colony selection.